We have characterized from cloned DNA the two nonallelic preproinsulin genes in the rat which differ in that one (gene I) lacks a 499 bp intron interrupting the coding region of the other (gene II). Since these genes are the products of a recent gene duplication, this intron was gained or lost since that time. To resolve this question we examined the chicken preproinsulin gene. We found that there is only one copy of this gene in the chicken genome. The structure of the chicken gene is similar to that of the rat gene II. Therefore, it represents the common ancestor. We conclude that introns can be lost during evolution. These studies gave us also the opportunity to carry out a detailed analysis of the molecular evolution of preproinsulin genes. We propose to define sequences in preproinsulin-gene associated DNA related to expression. For this purpose, in vitro modified gene-associated sequences will be assayed in an in vitro transcriptional system or in vivo, following microinjection. We have shown that microinjection of defined, cloned sequences, using iontophoresis, can be used to transform cultured cells. The injected sequences are stably integrated within the host genome. The integration frequency is extremely high, greater than 50 percent. Thus this new method we developed can be used to introduce any gene into any cell. Our future studies will also include, the characterization of preproinsulin genes in other organisms for evolutionary studies, a study of the structural organization of preproinsulin genes in diabetic animal models and a study of the mechanism of insulin biosynthesis stimulated by glucose.